Biochem. J. (2010) 432, 295–301 (Printed in Great Britain)
295
doi:10.1042/BJ20100996
Chelation of lysosomal iron protects against ionizing radiation
Carsten BERNDT*†1,2 , Tino KURZ‡1 , Markus SELENIUS§, Aristi P. FERNANDES§, Margareta R. EDGREN and Ulf T. BRUNK‡
*Division for Biochemistry, Department for Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden, †Institute for Clinical Cytobiology and
Cytopathology, Philipps-Universität, 35037 Marburg, Germany, ‡Division of Pharmacology, Faculty of Health Sciences, Linköping University 581 85 Linköping, Sweden, §Division of
Pathology, Department of Laboratory Medicine, Karolinska Institute, 141 86 Stockholm, Sweden, and Division of Medical Radiation Physics, Department of Oncology-Pathology,
Karolinska Institute, 171 76 Stockholm, Sweden
Ionizing radiation causes DNA damage and consequent apoptosis,
mainly due to the production of hydroxyl radicals (HO• )
that follows radiolytic splitting of water. However, superoxide
(O2 • − ) and H2 O2 also form and induce oxidative stress with
resulting LMP (lysosomal membrane permeabilization) arising
from iron-catalysed oxidative events. The latter will contribute
significantly to radiation-induced cell death and its degree largely
depends on the quantities of lysosomal redox-active iron present
as a consequence of autophagy and endocytosis of iron-rich
compounds. Therefore radiation sensitivity might be depressed
by lysosome-targeted iron chelators. In the present study, we
have shown that cells in culture are significantly protected from
ionizing radiation damage if initially exposed to the lipophilic
iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone),
and that this effect is based on SIH-dependent lysosomal
stabilization against oxidative stress. According to its doseresponse-modifying effect, SIH is a most powerful radioprotector
and a promising candidate for clinical application, mainly to
reduce the radiation sensitivity of normal tissue. We propose,
as an example, that inhalation of SIH before each irradiation
session by patients undergoing treatment for lung malignancies
would protect normally aerated lung tissue against life-threatening
pulmonary fibrosis, whereas the sensitivity of malignant lung
tumours, which usually are non-aerated, will not be affected by
inhaled SIH.
INTRODUCTION
In the lung alveoli exist a large number of macrophages,
many of which have engulfed erythrocytes and, consequently,
contain iron-rich lysosomes that may burst as a consequence of
ionizing radiation, induce macrophage death and contribute to
the induction of radiation pneumonitis and pulmonary fibrosis.
Furthermore, other pulmonary cell types may have iron-rich
lysosomes and, interestingly, the reparative autophagy that is
initiated by irradiation greatly enhances the amount of lysosomal
redox-active iron [7]. Reparative autophaphagy is a way for cells
to degrade damaged constituents and involves the breakdown of
cellular ferruginous materials, such as ferritin and mitochondria.
As a result, autophagolysosomes transiently become rich in lowmass redox-active iron, although it is eventually transported out of
the lysosomal compartment to be stored in ferritin, or exploited
in a variety of anabolic processes within mitochondria and the
cytosol [9,10].
Lung cancer is presently the leading cause for cancer-related
death worldwide [11]. Many cases of lung cancer require ionizing
radiation as part of the management of this common group of
diversified malignancies with a generally poor outcome. A major
problem that limits the dose of radiation is the risk of inducing
pulmonary fibrosis which may turn out to be life-threatening
[12]. Consequently, it is often necessary to apply a dose of
ionizing radiation that is less than optimal for effective therapy.
Improvement of therapeutic efficiency is therefore obviously
needed. In the present paper, we suggest a somewhat unorthodox
way of handling the situation. Most drugs or treatments aim
to enhance the irradiation efficiency on tumours, whereas we
instead suggest strategies for the protection of the surrounding
Non-surgical cancer therapy, e.g. chemo- and radio-therapy, is
mainly based on the induction of apoptotic cell death following the
production of ROS (reactive oxygen species). Proteins combating
oxidative stress, such as members of the thioredoxin family
of proteins, superoxide dismutases or catalases, are often upregulated in tumour cells and associated with resistance to such
therapies [1–5]. It is generally assumed that DNA damage,
mediated by hydroxyl radicals (HO• ) that are formed by radiolytic
cleavage of water, is responsible for cell death caused by ionizing
radiation [6].
It has been pointed out previously that, in addition to DNA
damage and resultant p53-mediated cell death, LMP (lysosomal
membrane permeabilization) induced by oxidative stress is a
contributing factor in apoptotic cell death caused by ionizing
radiation [7]. Such LMP is dependent on intralysosomal redoxactive iron that causes peroxidation and fragmentation of the
lysosomal membrane secondary to the oxidative stress that
radiation induces [7,8]. Fenton-type reactions between H2 O2 and
redox-active iron lead to the formation of HO• radicals inside the
lysosomal compartment. It therefore follows that the lysosomal
concentration of redox-active iron would be directly related to the
extent of LMP. It has been found that irradiation-induced LMP
can be abrogated by chelation of lysosomal redox-active iron
using DFO (desferrioxamine) [7]. DFO, however, stays within
the lysosomal compartment following its endocytic uptake, causes
iron starvation with ensuing cell death and is obviously not a wellsuited chelator.
Key words: ionizing radiation, iron chelation, lung cancer,
lysosome, oxidative stress, salicylaldehyde isonicotinoyl
hydrazone (SIH).
Abbreviations used: AO, Acridine Orange; DFO, desferrioxamine; HBSS, Hanks balanced salt solution; LMP, lysosomal membrane permeabilization;
PF, protection factor; ROS, reactive oxygen species; SIH, salicylaldehyde isonicotinoyl hydrazone.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
296
C. Berndt and others
normal tissue. So far, only one radioprotector, amifostine, which
incidentally also happens to be a lysosomotropic iron chelator,
has been explored in clinical trials [13–15].
In the present study, we have assessed the effect of lysosomal
iron chelation by the lipophilic chelator SIH (salicylaldehyde
isonicotinoyl hydrazone) on cell survival following irradiation in a
variety of cultured cells. Since SIH enters and leaves cells rapidly,
being in equilibrium with its concentration in the surrounding
medium, it can easily be rinsed away and, in contrast with DFO,
has no long-lasting effects [16].
Following irradiation, the cultures were incubated for 10–
14 days, with a change of medium after 5–7 days. Thereafter,
colonies were fixed, stained and counted. Radiation-survival
curves were constructed from one to four independent
experiments.
Dose–response models for clonogenic cell survival
The LQ (linear quadratic) model [19] was used to fit data with the
least-squares method, where the probability for clonogenic cell
survival S at a dose D is given by [20]:
EXPERIMENTAL
S = exp(−α D − β D 2 )
Chemicals
The doses for 10 % survival levels were calculated to estimate the
dose-modifying fraction, in this case the PF (protection factor).
AO (Acridine Orange) base was from Gurr. SIH (a gift from
Professor Des Richardson, University of Sydney, Sydney, New
South Wales, Australia) was dissolved in DMSO and then diluted
in ethanol in such a way that the final stock solution contained
SIH at a concentration of 10 mM in a 10 % DMSO/90 % ethanol
vehicle. Aliquots of this stock solution were added to cell culture
medium to obtain final concentrations of 10–100 μM SIH. Since
DMSO is a well-known scavenger of HO• radicals, and protects
against ionizing radiation [17], initial experiments were carried
out to ensure that the low final concentration of the DMSO/ethanol
vehicle had no influence on the cellular sensitivity to radiation or
H2 O2 (results not shown). All other chemicals were from Sigma–
Aldrich.
Cell cultures
Cell lines were originally from the A.T.C.C. (Manassas, VA,
U.S.A.) or Uppsala University. HeLa and J774 cells were grown in
DMEM (Dulbecco’s modified Eagle’s medium) (Gibco), U1690
cells were grown in MEM (minimal essential medium), and the
cell lines U2020, U1810 and U1906e were grown in RPMI
1640 (Gibco). All media were supplemented with 10 % (v/v)
heat-inactivated FBS (fetal bovine serum), 2 mM glutamine and
100 units · ml − 1 penicillin/streptomycin (PAA). Cells were grown
in plastic flasks and 35-mm-diameter Petri dishes (Corning) at
37 ◦ C in a 90 % humidified atmosphere containing 5 % CO2 .
They were subcultivated once or twice a week.
Ionizing radiation
γ -I radiation was performed with a 137 Cs source (Scanditronix)
at the Karolinska Institute, Stockholm, at a photon dose rate of
0.5 Gy · min − 1 . Dosimetry was performed using an ionization
chamber as well as with ferrous sulfate. According to the
sensitivity of the cell lines used, doses were in the range 0–8 Gy.
Cells were transported in insulated boxes and irradiated at room
temperature (22 ◦ C). The irradiation was carried out in fresh
medium, with or without SIH. When applied, SIH was added
30 min before irradiation. The irradiated medium was replaced
by fresh growth medium (without SIH) when cells were returned
to standard culture conditions.
Estimation of growth curves
Survival of HeLa cells was estimated as described above. Cultures
were prepared in numbers that allowed daily counting for 3 days
following irradiation. An alternative method to measure cell
survival following irradiation was applied to the cell lines U1906e
and U1810 as these cell types do not readily form colonies. In
those cases, cells were seeded and grown in 25 cm2 culture flasks
for 24 h before irradiation that was performed under conditions
described above. Cells were then routinely subcultured in a 1:4
ratio and counted three times during a period of 14–16 days
following irradiation. Estimation of cell numbers (cells/ml) was
obtained by assaying attenuance (D) at 600 nm on trypsinized
single-cell suspensions. The D600 values were compared with a
standard curve that was constructed previously by counting
a series of diluted cell suspensions in a Bürker chamber. Finally,
growth curves were obtained by comparing cell numbers at a
number of time points in relation to the cell number at the previous
subcultivation.
Cell survival following exposure to H2 O2
Cells were seeded in 96-well plates at 104 cells/well. After 16 h,
the cells were incubated for 1 h with different concentrations
of H2 O2 (0–100 mM) in HBSS (Hanks balanced salt solution)
with or without 100 μM SIH present. Some cells were
incubated with 30 μM FeCl3 for 5 h before the H2 O2 treatment
[when added to culture medium, Fe(III) forms insoluble iron
phosphates/hydroxides that are taken up by endocytosis and
transported to the lysosomal compartment]. Following 1 h of
H2 O2 exposure (during this period of time, most of the H2 O2
was degraded by the cells) cells were washed and returned
to standard culture conditions. The number of viable cells
was determined 24 h later using the Cell Proliferation Kit II
(Roche Applied Science). This assay is based on formation
of a coloured formazan following mitochondrial oxidation of
the tetrazolium salt XTT [2,3-bis-(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide] by metabolically
active cells. The dye was quantified using a microplate reader
(SpectraMax 340PC, Molecular Devices) at 490 and 650 nm.
Estimation of clonogenic cell survival
Appropriate cell numbers were plated for survival using the
clonogenic assay technique described previously [18]. Single-cell
suspensions were plated in 35-mm-diameter plastic Petri dishes
or six-well plates in triplicate or quadruplicate in a final medium
volume of 3 ml/dish or well and then left in the incubator for
3–4 h to attach before irradiation, which was performed as
described above.
c The Authors Journal compilation c 2010 Biochemical Society
Lysosomal membrane stability assay
AO is a metachromatic fluorophore and a lysosomotropic base
(pK a = 10.3), which becomes charged (AOH+ ) and retained by
proton trapping within acidic compartments, mainly secondary
lysosomes (pH 4.5–5.5). Using blue light excitation, normal cells
show bright red lysosomes (indicating high AO concentration) and
weak green cytoplasmic and nuclear fluorescence (indicating low
Iron and ionizing radiation
Figure 1
297
The iron chelator SIH preserves cell growth following ionizing radiation
HeLa (A), U1906e (B), and U1810 (C and D) cells were seeded approx. 14 h before being exposed for 30–60 min to 10 μM SIH (diamonds) or not (circles) followed by irradiation (closed symbols,
continuous lines) with 2 (B), 3 (A) or 5 Gy (C and D) or without irradiation (open symbols, broken lines). (D) Survival of U1810 cells 2 weeks following irradiation under the protection of 10 μM
SIH (black bar) or without such protection (white bar) compared with non-irradiated cells. Statistical significance for the SIH-mediated protection against radiation was calculated using Student’s t
test (***P < 0.001, **P < 0.01, *P < 0.05).
Figure 2
The iron chelator SIH protects cells against ionizing-radiation-induced cell death
HeLa (A and B), J774 (C) and U1690 (D) cells were seeded approx. 14 h before being incubated without (circles) or with 10 μM (diamonds) or 20 μM (squares) SIH for 30–60 min and then
irradiated with doses between 1 and 8 Gy. The surviving fractions, based on the number of colonies found 10–14 days following the irradiation, were determined and plotted against the radiation
dose. (B) Enhanced levels of lysosomal iron (a result of endocytotic uptake of iron phosphate/hydroxide) increase the damaging effect of ionizing radiation.
AO concentration). The AO relocation technique [16,21] was used
to show early lysosomal damage. The lysosomes of cells are preloaded with AO before exposure to any treatment that is supposed
to cause LMP, which is registered by flow cytofluorimetry as an
increase in green AO fluorescence that results from AO relocation
to the cytoplasm.
Approx. 106 U1690 cells in 2 ml of complete medium were
exposed to 10 μg/ml AO for 15 min under otherwise standard
conditions. Cells were then washed with complete medium and
equilibrated under standard conditions for another 15 min, before
they were exposed to 100 μM H2 O2 in HBSS, with or without
100 μM SIH, for 30 min at 37 ◦ C. At the end of the oxidative
stress period, cells were kept under standard culture conditions
for another 30 min before they were trypsinized, and green AO
fluorescence was analysed by flow cytofluorimetry (FACScan,
Becton-Dickinson) using the FL1 channel.
RESULTS
Iron chelation protects cells against radiation-induced cell death
In order to find out whether iron chelation protects against cell
death, several cell lines were irradiated with or without the iron
chelator SIH present. We tested the mouse macrophage cell line
J774, the cervical cancer cell line HeLa, and a number of lung
cancer cell lines, U1690, U1906e and U1810. U1690 is a smallcell lung cancer cell line [22], U1810 is a radioresistant non-smallcell lung cancer cell line [23], and U1906e is a radiosensitive
small-cell lung cancer subcell line [24]. First, growth curves
of HeLa, U1906e and U1810 were recorded (Figure 1). Cells
were exposed to a single fraction of ionizing radiation at 2 Gy
(Figure 1B), 3 Gy (Figure 1A) or 5 Gy (Figures 1C and 1D)
with or without 10 μM SIH. In HeLa cells, we investigated
the direct effect of radiation on cell survival, and in the lung
cancer cell lines, we investigated the ability to repopulate after
irradiation. Both immediate protection and repopulation were
significantly improved by SIH. SIH-treated non-irradiated cells
grew better than control cells (Figure 1). Since DMSO is known
as a potent scavenger of HO• radicals [17], we ensured that DMSO
in the 0.01–0.1 % range had no protective effect (results not
shown). Next, we determined the surviving fractions based on
the clonogenic cell survival assay using HeLa, J774 and U1690
cells (Figure 2). The ability to undergo five or more cell divisions
following irradiation is used as an indication of cell survival. A
survivor that has retained its reproductive integrity and is able
to proliferate continuously to produce a large clone or colony
is said to be clonogenic. SIH increased the surviving fractions
in all cell lines studied. In line with this result, exposure to
an Fe(III) phosphate/hydroxide precipitate (obtained by adding
10 μM FeCl3 to the medium) that was endocytosed by the
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298
Figure 3
C. Berndt and others
The iron chelator SIH protects U2020 and U1690 cells against H2 O2 -induced cell death
U2020 (A) and U1690 (B) cells were seeded in 96-well-plates (104 cells/well), whereas U1690 cells were also seeded in six-well plates at a density of 100–800 cells/well (C). The cells were then
exposed for 1 h to H2 O2 (initially at 0–100 mM) without (open squares) or together with (closed circles) 100 μM SIH. Cell viability (A and B) and clonogenic cell survival (C) were calculated 24 h
and 10–14 days after return to standard culture conditions. The reduced cell survival following the addition of 30 μM FeCl3 to the medium 5 h before exposure to H2 O2 is shown by the broken line
with filled diamonds (B). (C) Survival of cells exposed to H2 O2 in the presence (black bars) of 20 μM SIH or not (white bars). Note that unprotected cells did not survive the H2 O2 exposure. Results
are means +
− S. D. of four to six repeats for two independent experiments.
Table 1 PFs (or survival indices) for various cell lines following ionizing
radiation in the presence of the iron chelator SIH
PFs were calculated based on clonogenic cell survival assays (see Figure 2) as the ratio of the
doses with and without SIH given 10 % survival.
Cell type
SIH concentration (μM)
PF
Additional radiation dose without
changes in cell survival (%)
HeLa
HeLa
J774
U1690
10
20
10
20
1.20
1.78
1.30
1.20
20
78
30
20
cells for 4 h before irradiation decreased the surviving fractions
(Figure 2B). Compared with the control cells (irradiated without
prior iron exposure), only approx. 30 % of the iron-loaded cells
survived the radiation doses of 6 Gy (Figure 2B) and 8 Gy
(results not shown). Protection of cells against radiation was partly
dependent on the SIH concentration. The PFs were calculated as
the ratio of the doses that gave 10 % survival with and without
SIH protection respectively. As shown in Table 1, the PF for
HeLa cells increased from 1.20 to 1.78 following doubling of
the SIH concentration from 10 to 20 μM. This means that, as
a consequence of SIH protection, the radiation doses can be
increased by 20 and 80 % respectively without a change in the
survival rate. The PF for J774 cells was 1.30 at 10 μM SIH,
whereas it was 1.20 for U1690 at 20 μM SIH. When cells were
exposed to 2 and 4 Gy, which are reasonable daily doses in the
treatment of lung cancers, the PF for U1690 was found to be
between 1.40 and 1.80 in the presence of 20 μM SIH.
previously for ionizing radiation (Figure 2B), survival decreased
if cells were exposed to an iron phosphate complex before the
induction of oxidative stress (Figure 3B). The EC50 value for
U1690 cells fell to 53 μM following incubation with 30 μM FeCl3
for 5 h before ensuing H2 O2 treatment. It should be pointed out that
the addition of FeCl3 to culture medium results in the formation
of an iron phosphate/hydroxide precipitate that is endocytosed by
the cells. The lysosomal compartment is thereby enriched with
iron.
Using again the clonogenic cell-survival assay, we calculated
how many U1690 cells survived exposure to different
concentrations of H2 O2 compared with untreated cells. We found
that, without SIH protection, no U1690 cells survived the 1 h
period of H2 O2 exposure at 50–150 μM initially, whereas 50–
100 % of the cells that were protected by 20 μM SIH did so
(Figure 3C).
Iron chelation influences lysosomal stability under conditions of
oxidative stress
To obtain further insights into the protection mechanism afforded
by the iron chelator SIH, we assayed LMP, given the fact that
redox-active iron is mainly found inside lysosomes [21,27–30].
U1690 cells were subjected to the AO-relocation test. Following
AO loading, cells were exposed for 30 min to 100 μM H2 O2 with
or without 100 μM SIH in HBSS, and green fluorescence was
assayed by flow cytofluorimetry after another 30 min (Figure 4A).
Compared with the control cells, the mean green fluorescence
increased up to 156 % following exposure to H2 O2 only, whereas
cells exposed to 100 μM H2 O2 under the protection of 100 μM
SIH showed only a small increase in the mean green fluorescence;
up to 110 % of the control cells (Figure 4B).
Iron chelation protects cells against H2 O2 -induced cell death
Since it is believed that the effect of ionizing radiation partly
depends on intracellular formation of H2 O2 [25] with ensuing
LMP [7], we investigated protection by SIH against H2 O2 -induced
cell death. The small-cell lung cancer lines U2020 [26] and U1690
were exposed to various concentrations of H2 O2 with or without
100 μM SIH present (Figures 3A and 3B) and cell survival
was calculated 24 h later. For U2020 cells, the EC50 H2 O2 value
increased from 0.22 mM to 7.85 mM (Figure 3A) and for the
U1690 cells from 93 μM to 580 μM (Figure 3B). As was found
c The Authors Journal compilation c 2010 Biochemical Society
DISCUSSION
Apart from radiolytic cleavage of water leading to formation of
HO• radicals, the simultaneous production of H2 O2 is a wellknown effect of exposure of tissues to ionizing radiation [7,25].
However, the possible influence of H2 O2 on radiation-induced
cellular damage does not usually seem to be fully taken into
account. This is somewhat surprising, since in a paper from 1962,
Otto Warburg pointed out that the cellular effects of exposure to
Iron and ionizing radiation
299
Figure 4 SIH-protection against the effect of H2 O2 is a function of lysosomal
stabilization
(A) U1690 cells were pre-loaded with AO 16 h after seeding. Cells were then exposed for
30 min to initially 100 μM H2 O2 in HBSS in the presence of (black histogram) or without (clear
histogram) 100 μM SIH, or just kept in HBSS (grey histogram). After another 30 min under
standard culture conditions, the cells were trypsinized, and green FL1 fluorescence, being an
indicator of AO relocated to the cytosol as a result of lysosomal rupture, was analysed by flow
cytofluorimetry. (B) Protection by SIH of lysosomes against H2 O2 (Student’s t test, ***P <
0.001). White bar, cells exposed to 100 μM H2 O2 only; black bar, cells exposed to 100 μM
H2 O2 in the presence of 100 μM SIH.
ionizing radiation or to H2 O2 show substantial similarities [31]:
H2 O + ionizing radiation → HO• + H+ + e−
O2 + e− → O•−
2
•−
+
O•−
2 + O2 + 2H → H2 O2 + O2
Following studies of the damaging effects of randomly formed
HO• radicals, it has been postulated that these short-lived (10 − 9 s)
and extremely aggressive radicals react with nuclear DNA on the
very spot where they are formed, causing adducts, mutations and
single- and double-strand breaks with resulting cellular damage.
Even if it is not definitively proven that HO• radical-induced DNA
damage is the main cause of cellular injury following irradiation,
there is an overwhelming amount of indirect evidence that this is
indeed the case, and there seems to be little reason to question
this dogma. However, apart from radiolytic cleavage of water,
HO• radicals can also be produced by Fenton-type (transitionmetal-mediated) reactions, which gives an incentive to examine
the occurrence of such reactions during ionizing radiation:
Fe2+ + H2 O2 → Fe3+ + HO• + OH−
2+
Fe3+ + O•−
+ O2
2 → Fe
Obviously, the presence of redox-active iron in direct contact
with DNA would give rise to massive site-specific Fentontype chemistry, given the radiation-induced presence of H2 O2
and superoxide (O2 • − ). Under normal conditions, there are no
indications of any significant amount of low-mass redox-active
iron that is in juxtaposition to DNA [32–34]. However, as has been
demonstrated, under conditions of oxidative stress, lysosomal
rupture will occur, iron will be relocated and DNA damage
initiated [7,32–34].
Because the lysosomal compartment is the centre for normal
autophagic turnover of all organelles and most long-lived proteins,
many of which are ferruginous compounds, lysosomes of
all cells contain low-mass redox-active iron, explaining their
vulnerability to oxidative stress [9,10]. An additional way of
Figure 5 Schematic representation of suggested mechanisms behind SIHmediated protection against radiation-induced cell death
This scenario is based on the fact that lung tumours are non-aerated and therefore not reached
by inhaled SIH, whereas normal lung tissue is aerated and exposed to SIH. Consequently,
the normal tissue will be protected against cell death induced by iron-mediated Fenton-type
reactions following ionizing radiation (IR). If so, a substantially larger dose of irradiation could
be applied over a pulmonary field without induction of dangerous fibrosis (for details, see the
text).
loading lysososomes with iron is of importance when scavenger
cells, e.g. alveolar macrophages, endocytose erythrocytes and
thereby enrich their lysosomal compartment with redox-active
iron. The lysosomal compartment is acidic and rich in reducing
equivalents, such as cysteine and glutathione, ensuring that any
low-mass iron present would largely be in Fe2+ form [8,35]. That
in turn would promote the generation of HO• radicals from H2 O2
diffusing into this compartment.
Lysosomes show widely different sensitivity to oxidative stress
[36]. Using vital staining with lysosomotropic fluorochromes, e.g.
AO or other available lysotrackers, it was found that, after heavy
oxidative stress, some lysosomes always remain intact, while even
low oxidative stress results in the rupture of a small, but obviously
very sensitive, population of lysosomes [36]. The explanation for
this phenomenon is probably that lysosomes that are actively
engaged in degradation of iron-containg macromolecules are rich
in iron, whereas resting lysosomes may contain little or nothing
of this transition metal [37].
Since the H2 O2 that forms throughout the cell during irradiation
is highly diffusible, it will enter the lysosomal compartment,
meet redox-active iron and induce violent Fenton-type reactions
with resultant LMP and release of lysosomal contents to the
surrounding cytosol (Figure 5). LMP will thus allow not only
the escape of low-mass iron from lysosomes, but also the
relocation of potent lysosomal cathepsins. Dependent on the
magnitude of lysosomal rupture, cell proliferation is stimulated or
arrested by a minor or a somewhat more pronounced lysosomal
destabilization respectively, whereas apoptosis or necrosis
has been found to follow moderate or major destabilization
respectively [37,38]. Consequently, the amelioration of LMP by
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300
C. Berndt and others
chelating lysosomal redox-active iron in a non-redox-active form
ought to reduce radiation sensitivity.
This hypothesis was supported previously by findings following
treatment with DFO at high doses for several hours before
irradiation [7]. Unfortunately, this hydrophilic and highmolecular-mass drug has the disadvantage of being taken up only
by endocytosis [39,40] and is retained in lysosomes where it
causes iron-starvation and, ultimately, cell death [9,10]. Therefore
DFO is not an ideal iron chelator for cellular protection against
oxidative stress. In the present study, we tested the radioprotective
effect of the lipophilic iron chelator SIH that is rapidly distributed
throughout the cell, but can also easily be washed away [16]. That
the protective effect reported in the present paper is due to the ironchelating effect of SIH is supported by the experiments showing
that addition of iron had a sensitizing effect (Figures 2B and 3B).
Although SIH has been shown to give excellent protection from
H2 O2 -induced oxidative stress [10,16], the findings of the present
study suggest that SIH also can be used to protect normal tissues
from radiation damage and may allow exposure to a higher than
normal dose of ionizing radiation without causing damage in the
normal tissue that is adjacent to a malignancy.
As an example, in the specific case of lung cancers, the tendency
of normal lung tissue to develop radiation-induced pulmonary
fibrosis severely limits the use of radiotherapy. Lung cancers
usually compress a branch of the bronchus system, leaving most
of the lung aerated, whereas the tumour itself and the lung tissue
distal to it are not (Figure 5). An aerosol containing a powerful
iron chelator might therefore protect normal lung tissue against
radiation, while the tumour itself should not be affected (Figure 5).
Our findings indicate that even low concentrations of SIH (10
or 20 μM) would allow the radiation dose to be increased by
80 % without the induction of additional damage to normal tissue.
This dose-modifying effect makes SIH one of the most powerful
radioprotectors tested so far. Interestingly, cells exposed to SIH
only actually grew better than the control cells, suggesting that
SIH protects against damage caused by having cells outside the
incubator. Inasmuch as SIH can be removed readily, allowing
high concentrations to be used, one might expect striking effects.
Indeed, SIH at 100 μM protected between 6- and 35-fold against
H2 O2 -induced cell death (Figure 3). Moreover, doubling the
SIH concentration increased its radiation-dose-modifying effect
4-fold (Table 1). All other radioprotective substances, e.g. thiol
(sulfhydryl) compounds, phytochemicals and aminothiols, which
are the most effective of the presently known radioprotectors, must
be applied in much higher concentrations (0.5–10 mM) in order
to reach similar PFs [41–47]. To confirm the high effectiveness
of SIH, and to compare it with other radioprotectors, animal
experiments are needed.
The additive effects of LMP, a consequence of intralysosomal
Fenton-type reactions secondary to enhanced cellular amounts of
H2 O2 , on top of the effects induced by direct formation of HO•
radicals following radiolytic cleavage of water, are dependent
on the presence of oxygen that allows formation of O2 • − and
H2 O2 (see the formulae at the beginning of the Discussion).
The importance of this additive effect is illustrated by the wellknown fact that hypoxic malignancies, e.g. those that infiltrate
bone tissue, respond less well to ionizing radiation. In hypoxic
tissues, there will be limited formation of O2 • − and H2 O2 and,
consequently, little LMP will take place.
In the present paper, we propose a new strategy for protection
of cells against ionizing radiation and explain its underlying
molecular mechanisms. Our results indicate that application of
SIH as an aerosol before each irradiation session would allow
exposure to a higher than normal irradiation dose and may increase
the survival chance for lung cancer patients, which now show
c The Authors Journal compilation c 2010 Biochemical Society
the highest mortality of all cancer patients [11], by protecting
normally aerated, and therefore accessible to an aerosol, lung
tissue, but not the solid malignancy without airways.
AUTHOR CONTRIBUTION
Carsten Berndt, Tino Kurz and Ulf Brunk wrote the manuscript; Carsten Berndt, Tino Kurz,
Aristi Fernandes, Margareta Edgren and Ulf Brunk designed the experiments; Carsten
Berndt, Tino Kurz, Markus Selenius and Margareta Edgren performed the experiments.
ACKNOWLEDGEMENTS
We thank Professor John Eaton, University of Louisville, Louisville, KY, USA, for valuable
suggestions.
FUNDING
We thank the Deutsche Forschungsgemeinschaft [grant number BE3259–2 (to C.B.)], the
Karolinska Institute (to C.B. and A.F.), Cancer och Allergifonden (to A.F.), Radiumhemmets
Forskningsfonder (to A.F.), and Hjärt-Lungfonden (to M.S.) for financial support.
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Received 7 July 2010/14 September 2010; accepted 16 September 2010
Published as BJ Immediate Publication 16 September 2010, doi:10.1042/BJ20100996
c The Authors Journal compilation c 2010 Biochemical Society